An essential element of most confocal systems is the pinhole. In particular a scanning confocal microscope has a single pinhole that is used to exclude out-of-focus light from the image. The size of the required pinhole is a function of the objective used on the microscope as well as the particular needs of the sample. For example, a user may use a larger pinhole to sacrifice optical sectioning for greater signal. There is then a need for means for changing the size of the pinhole for a confocal microscope.
The size of the pinhole needed is often determined in relation to the diffraction limited spot created by the signal. The diffraction limited spot forms an Airy disk in a plane at the focal point. The equation d=1.22λ/sin θ represents the approximate diameter (d) of the pinhole to give an ideal confocal signal for a given wavelength of light (λ) for an optical signal that focuses the light with angle (θ). This is the diameter where the whole first Airy disk can pass through the pinhole but none of the higher order rings. Increasing the diameter would increase the signal, but would allow more out-of-focus light.
The pinhole needed for most microscope setups has a diameter on the order of microns. The alignment of the pinhole to focal point of the signal is then critical and must have sub-micron repeatability and resolution. Recently there has been the introduction of compact mechanical manipulators that have nanometer resolution. A piezo stage is an example of such a system.
Consider two pinholes next to each other. Let the signal of interest be focused through one of the pinholes. The maximum signal that could “leak” into the neighboring pinhole can be approximated using the principle that equal intensities of light are found at all radii from the central spot. That is, a ring of 1 micron thickness at 10 microns away from the center would have approximately the same intensity of light as the central 1 micron spot. The area of such a ring would be A=2πrd, with the area of one pinhole P=πd2, making the ratio of the intensity out of the neighboring pinhole to the intensity out of the first pinhole d/2r. For our example the intensity ratio would be 5%. Now if we move the second pinhole away by 1 mm, then the ratio changes to 0.05%. In reality, the ratio would be much lower, because this approximation assumes an evenly illuminated volume, when in the real case, there will be lower contributions from out-of-focus areas. Regardless, one can imagine spacing several pinholes of various sizes within a few millimeters without much of a loss of confocality. One could then use the piezo stage to select between the pinholes. Even more ideally, one could make a two dimensional pattern of pinholes and use an XY stage to select the pinhole.
Exemplary embodiments as discussed herein could also be used to switch pinholes for a spatial filter system. A spatial filter makes use of a pinhole to clean up a laser beam. By rejecting the light that is outside of the first Airy disk, the resultant beam has a better Gaussian profile. Different pinholes sizes would allow one to balance intensity vs. beam quality.
The lenses used to re-collimate the light could be motorized or moved in a fashion such that the same device could be used to provide different amounts of beam expansion or contraction with no additional loss. Also, by slightly de-collimating the light, one would change the focal plane of the light's focus at the sample. This would cause spherical aberration to be introduced by the objective. This spherical aberration could be selected such that it corrects for the spherical aberration in the sample. Thus by causing the excitation light to converge or diverge, one could add spherical aberration correction to the device with no additional loss of light.